Calcofluor antifungal action depends on chitin and a functional high-osmolarity glycerol response (HOG) pathway: evidence for a physiological role of the Saccharomyces cerevisiae HOG pathway under noninducing conditions

Abstract

We have isolated several Saccharomyces cerevisiae mutants resistant to calcofluor that contain mutations in the PBS2 or HOG1 genes, which encode the mitogen-activated protein kinase (MAPK) and MAP kinases, respectively, of the high-osmolarity glycerol response (HOG) pathway. We report that blockage of either of the two activation branches of the pathway, namely, SHO1 and SLN1, leads to partial resistance to calcofluor, while simultaneous disruption significantly increases resistance. However, chitin biosynthesis is independent of the HOG pathway. Calcofluor treatment also induces an increase in salt tolerance and glycerol accumulation, although no activation of the HOG pathway is detected. Our results indicate that the antifungal effect of calcofluor depends on its binding to cell wall chitin but also on the presence of a functional HOG pathway. Characterization of one of the mutants isolated, pbs2-14, revealed that resistance to calcofluor and HOG-dependent osmoadaptation are two different physiological processes. Sensitivity to calcofluor depends on the constitutive functionality of the HOG pathway; when this is altered, the cells become calcofluor resistant but also show very low levels of basal salt tolerance. Characterization of some multicopy suppressors of the calcofluor resistance phenotype indicated that constitutive HOG functionality participates in the maintenance of cell wall architecture, a conclusion supported by the antagonism observed between the protein kinase and HOG signal transduction pathways.

FIG. 1

S. cerevisiae growth on plates supplemented with different calcofluor concentrations. Serial dilutions of each strain as indicated above the panels were plated onto complete SD medium supplemented with different calcofluor concentrations as indicated below the panels, incubated for 48 h at 28°C, and photographed. Note the different genetic backgrounds used: W303 (upper) and TM141 (lower). wt, wild type.

FIG. 2

Osmotic tolerance of different S. cerevisiae strains. (A) Osmotic tolerance of a logarithmically growing culture after incubation in the presence of 0.075 mg of calcofluor/ml for the indicated times. (B) Osmotic tolerance after osmotic (0.4 M NaCl) treatment for the indicated times. Osmotic tolerance is expressed as the percentage of cells able to grow on dehydration medium compared to those growing on YEPD. See Materials and Methods for details. ○, W303 (wild type); ■, TC1 (chs3⁻; ▴, LJYE14 (pbs2-14).

FIG. 3

Molecular characterization of the PBS2-14 mutation. (A) Schematic representation of PBS2-14 PCR cloning. Primer pairs and the conserved protein kinase (PK) domain are indicated. The arrow shows the approximate position of the E14 mutation. (B) Sequence discrepancy between wild type and mutant; the deleted base is indicated by an arrow. The protein translation is shown on the right side of each picture. (C) C-terminal sequence of Pbs2 and Pbs2-14 proteins. (D) Immunodetection of Pbs2p and Pbs2-14p in Western blot using anti-Pbs2p antibodies (see Materials and Methods). The arrowheads indicate the Pbs2 protein that is absent in the pbs2 null mutant. The altered Pbs2-14 protein is indicated by an asterisk. WT, wild type.

FIG. 4

Mutant growth in different media after transformation with different plasmids. Serial dilutions of each strain were plated onto SD medium supplemented with 0.5 mg of calcofluor/ml or YEPD medium supplemented with 0.9 M NaCl. The plates were photographed after 48 h of growth at 28°C. The strains are indicated above each picture. The plasmids carried by each strain are indicated at the right. (A) Calcofluor and salt plates are shown (W303 background). (B) Only calcofluor plates are shown. Note that TM285, TM257, and TM310 strains are in the TM141 background.

FIG. 5

Effects of different multicopy suppressors on calcofluor resistance. Serial dilutions of each strain were plated onto SD selective medium supplemented with different calcofluor concentrations (shown at bottom). The recipient strains (top) were transformed with multicopy plasmids containing the genes shown on the left.

FIG. 6

Epistatic relationship between the PKC and HOG signal transduction pathways. (A) Serial dilutions of each strain (15Dau background) were plated onto YEPD medium, and replica plates were incubated at the indicated temperatures for 48 h. (B) Isogenic W3031A or LJY1 (pbs2::LEU2) strains were transformed with plasmid pNV7-MKK1^P386 in which the hyperactive form of the MKK1 gene is under the control of the GAL1 promoter (40). Serial dilutions of each strain were plated onto selective medium with either glucose or galactose as a carbon source. Growth was scored after 2 (glucose) or 4 (galactose) days of growth.